Compositions comprising EPA and obeticholic acid and methods of use thereof

ABSTRACT

In various embodiments, the present invention provides compositions comprising eicosapentaenoic acid or a derivative thereof and obeticholic acid or a derivative thereof. In other embodiments, the present invention provides methods of treating and/or preventing NASH and/or PBC comprising co-administering to a subject in need thereof eicosapentaenoic acid or a derivative thereof and obeticholic acid or a derivative thereof.

PRIORITY CLAIM

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/747,678, filed on Dec. 31, 2012, the entire contents of which areincorporated herein by reference and relied upon.

BACKGROUND

Nonalcoholic steatohepatitis (“NASH”) is characterized by fat,inflammation and damage in the liver in people who consume little or noalcohol. NASH can lead to liver cirrhosis. NASH tends to be diagnosed inoverweight or obese middle-aged people who often have elevated bloodlipid levels and diabetes or prediabetes. Primary biliary cirrhosis(“PBC”) is characterized by inflammation and damage to the bile ducts.PBC is believed to be an autoimmune condition, but is more common inpeople who have been exposed to chemicals or who have had an infection.However, the causes of NASH and PBC are not well understood. There areno specific approved therapies for NASH, and only one approved drug fortreatment of PBC. A need exists for improved treatments for NASH andPBC.

SUMMARY

In various embodiments, the present invention provides a compositioncomprising about 500 mg to about 4 g (e.g., about 900 mg to about 1.1 g)of eicosapentaenoic acid or derivative thereof (e.g. ethyleicosapentaenoate) and about 1 mg to about 500 mg of obeticholic acid ora derivative thereof (e.g., a salt, ester, an amino acid conjugate, or acombination thereof).

In various embodiments, the present invention provides a compositioncomprising at least about 60%, 70%, 80% or 90%, by weight of all fattyacids present, eicosapentaenoic acid or derivative thereof (e.g. ethyleicosapentaenoate) and about 1 mg to about 500 mg of obeticholic acid ora derivative thereof (e.g., a salt, ester, an amino acid conjugate, or acombination thereof).

In various embodiments, the present invention provides a method oftreating and/or preventing NASH or PBC in a subject or a subject group,the method comprising co-administering orally to the subject or subjectgroup a pharmaceutical composition comprising about 2 g to about 4 g ofeicosapentaenoic acid or derivative thereof (e.g. ethyleicosapentaenoate) and about 1 mg to about 500 mg of obeticholic acid orderivative thereof. In some embodiments, the eicosapentaenoic acid orderivative thereof (e.g. ethyl eicosapentaenoate) and the obeticholicacid or derivative thereof are formulated in a single dosage unit (i.e.in the same capsule, sachet, tablet, etc).

In various embodiments, the present invention provides a method oftreating and/or preventing NASH or PBC in a subject or a subject group,the method comprising co-administering orally to the subject or subjectgroup about 2 to about 4 capsules per day, each capsule comprising about900 mg to about 1.1 g of eicosapentaenoic acid or derivative thereof(e.g. ethyl eicosapentaenoate) and optionally not more than about 20%,by weight of all fatty acids present, docosahexaenoic acid or itsesters, and about 1 mg to about 500 mg of obeticholic acid or derivativethereof.

In various embodiments, the present invention provides a method ofinhibiting N. aromaticivorans in a mammal, the method comprisingco-administering orally to the mammal daily a pharmaceutical compositioncomprising about 2 g to about 4 g ethyl eicosapentaenoate and about 1 mgto about 500 mg of obeticholic acid or derivative thereof. In someembodiments, the ethyl eicosapentaenote and the obeticholic acid orderivative thereof are formulated in a single dosage unit. In someembodiments, the mammal is a human.

In various embodiments, the present invention provides a method ofinhibiting N. aromaticivorans in a mammal, the method comprisingco-administering orally to the mammal about 2 to about 4 capsules perday, each capsule comprising about 900 mg to about 1.1 g ofeicosapentaenoic acid or derivative thereof (e.g. ethyleicosapentaenoate) and optionally not more than about 20%, by weight ofall fatty acids present, docosahexaenoic acid or its esters, and about 1mg to about 500 mg of obeticholic acid or derivative thereof. In someembodiments, the mammal is a human.

In various embodiments, the present invention provides a method ofinhibiting Farnesoid X receptor (“FXR”) in a mammal, the methodcomprising co-administering orally to the mammal about 2 g to about 4 gper day of a pharmaceutical composition comprising at least about 90%,by weight of all fatty acids present, ethyl eicosapentaenoate and about1 mg to about 500 mg of obeticholic acid or derivative thereof. In someembodiments, the eicosapentaenoic acid or derivative thereof (e.g. ethyleicosapentaenoate) and the obeticholic acid or derivative thereof areformulated in a single dosage unit. In some embodiments, the mammal is ahuman.

In various embodiments, the present invention provides a method ofinhibiting FXR in a mammal, the method comprising co-administeringorally to the mammal about 2 to about 4 capsules per day, each capsulecomprising about 900 mg to about 1.1 g of eicosapentaenoic acid orderivative thereof (e.g. ethyl eicosapentaenoate) and not more thanabout 20%, by weight of all fatty acids present, docosahexaenoic acid orits esters, and about 1 mg to about 500 mg of obeticholic acid orderivative thereof. In some embodiments, the mammal is a human.

In one embodiment, a pharmaceutical composition useful in accordancewith the invention comprises, consists of or consists essentially of atleast 95% by weight ethyl eicosapentaenoate (EPA-E), about 0.2% to about0.5% by weight ethyl octadecatetraenoate (ODTA-E), about 0.05% to about0.25% by weight ethyl nonadecapentaenoate (NDPA-E), about 0.2% to about0.45% by weight ethyl arachidonate (AA-E), about 0.3% to about 0.5% byweight ethyl eicosatetraenoate (ETA-E), and about 0.05% to about 0.32%ethyl heneicosapentaenoate (HPA-E). In another embodiment, thecomposition is present in a capsule shell. In another embodiment, thecomposition contains substantially no or no amount of docosahexaenoicacid (DHA) or derivative thereof such as ethyl-DHA (DHA-E).

In another embodiment, the invention provides a method of treatingand/or preventing NASH and/or PBC and further treating moderate tosevere hypertriglyceridemia comprising administering a composition asdescribed herein to a subject in need thereof.

In any of the foregoing embodiments, the eicosapentaenoic acid orderivative thereof (e.g. ethyl eicosapentaenoate) and obeticholic acidor derivative thereof can be co-formulated as a single dosage unit orcan be formulated as two to a plurality of dosage units for coordinated,combination or concomitant administration.

These and other embodiments of the present invention will be disclosedin further detail herein below.

DETAILED DESCRIPTION

While the present invention is capable of being embodied in variousforms, the description below of several embodiments is made with theunderstanding that the present disclosure is to be considered as anexemplification of the invention, and is not intended to limit theinvention to the specific embodiments illustrated. Headings are providedfor convenience only and are not to be construed to limit the inventionin any manner. Embodiments illustrated under any heading may be combinedwith embodiments illustrated under any other heading.

The use of numerical values in the various quantitative values specifiedin this application, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges were both preceded by the word “about.” Also, thedisclosure of ranges is intended as a continuous range including everyvalue between the minimum and maximum values recited as well as anyranges that can be formed by such values. Also disclosed herein are anyand all ratios (and ranges of any such ratios) that can be formed bydividing a disclosed numeric value into any other disclosed numericvalue. Accordingly, the skilled person will appreciate that many suchratios, ranges, and ranges of ratios can be unambiguously derived fromthe numerical values presented herein and in all instances such ratios,ranges, and ranges of ratios represent various embodiments of thepresent invention.

The term “treatment” in relation a given disease or disorder, includes,but is not limited to, inhibiting the disease or disorder, for example,arresting the development of the disease or disorder; relieving thedisease or disorder, for example, causing regression of the disease ordisorder; or relieving a condition caused by or resulting from thedisease or disorder, for example, relieving, preventing or treatingsymptoms of the disease or disorder. The term “prevention” in relationto a given disease or disorder means: preventing the onset of diseasedevelopment if none had occurred, preventing the disease or disorderfrom occurring in a subject that may be predisposed to the disorder ordisease but has not yet been diagnosed as having the disorder ordisease, and/or preventing further disease/disorder development ifalready present.

In one embodiment, the invention provides a composition comprising about500 mg to about 4 g of eicosapentaenoic acid or derivative thereof (e.g.ethyl eicosapentaenoate)and less than 20%, by weight of all fatty acidspresent, docosahexaenoate or its esters, and about 1 mg to about 500 mgof obeticholic acid or a derivative thereof.

In various embodiments, the invention provides a composition comprisingat least about 60%, about 70%, about 80% or about 90%, by weight of allfatty acids present, eicosapentaenoic acid or derivative thereof (e.g.ethyl eicosapentaenoate) and about 1 mg to about 500 mg of obeticholicacid or a derivative thereof.

In one embodiment, the present invention provides a method of treatingand/or preventing NASH and/or PBC, the method comprising administeringto a subject or subject group in need thereof a pharmaceuticalcomposition as described herein. In some embodiments, the subject orsubject group has one or more of: an elevated baseline alanineaminotransferase (“ALT”) level, an elevated baseline aspartateaminotransferase (“AST”) level, liver fibrosis, an elevated baselinegamma-glutanyl transferase level, an elevated baseline alkalinephosphatase level, an elevated baseline antimitochondrial antibodylevel, an elevated baseline antinuclear antibody level, an elevatedtotal serum bilirubin level, and an elevated transaminase level. In someembodiments, the invention provides a method of treating and/orpreventing NASH and/or PBC in a subject or a subject group, the methodcomprising co-administering orally to the subject or subject group about2 g to about 4 g per day of a pharmaceutical composition comprising atleast about 60%, 70%, 80% or 90%, by weight of all fatty acids present,eicosapentaenoic acid or derivative thereof (e.g. ethyleicosapentaenoate) and about 1 mg to about 500 mg of obeticholic acid orderivative thereof. In some embodiments, the eicosapentaenoic acid orderivative thereof (e.g. ethyl eicosapentaenoate) and the obeticholicacid or derivative thereof are formulated in a single dosage unit. Insome embodiments, the method comprises co-administering orally to thesubject or subject group about 2 to about 4 capsules per day, eachcapsule comprising about 900 mg to about 1.1 g of eicosapentaenoic acidor derivative thereof (e.g. ethyl eicosapentaenoate) and not more thanabout 20%, by weight of all fatty acids present, docosahexaenoic acid orits esters, and about 1 mg to about 500 mg of obeticholic acid orderivative thereof.

Novosphingobium aromaticivorans is a gram-negative alphabacterium knownto be strongly associated with PBC occurrence. The mechanism is not wellunderstood, but appears to involve a cross reaction between the proteinsof the bacterium and mitochondrial proteins of the liver cells. The geneencoding CD101 (cluster of differentiation 101, also referred to asimmunoglobulin superfamily, member 2 or IGSF2) may also play a role inhost susceptibility to PBC. In one embodiment, the present inventionprovides a method of inhibiting N. aromaticivorans in a mammal, themethod comprising administering to mammal a pharmaceutical compositionas described herein. In some embodiments, the mammal has one or more of:an elevated baseline alanine aminotransferase (“ALT”) level, an elevatedbaseline aspartate aminotransferase (“AST”) level, liver fibrosis, anelevated baseline gamma-glutanyl transferase level, an elevated baselinealkaline phosphatase level, an elevated baseline antimitochondrialantibody level, an elevated baseline antinuclear antibody level, anelevated total serum bilirubin level, and an elevated transaminaselevel. In some embodiments, the present invention provides a method ofinhibiting N. aromaticivorans in a mammal, the method comprisingco-administering orally to the mammal about 2 g to about 4 g per day ofa pharmaceutical composition comprising at least about 90%, by weight ofall fatty acids present, ethyl eicosapentaenoate and about 1 mg to about500 mg of obeticholic acid or derivative thereof. In some embodiments,the method comprises co-administering orally to the mammal about 2 toabout 4 capsules per day, each capsule comprising about 900 mg to about1 g of ethyl eicosapentaenoate and not more than about 20%, by weight ofall fatty acids present, docosahexaenoic acid or its esters, and about 1mg to about 500 mg of obeticholic acid or derivative thereof. In someembodiments, the ethyl eicosapentaenote and the obeticholic acid orderivative thereof are formulated in a single dosage unit. In someembodiments, the mammal is a human.

Farnesoid X receptors (“FXR”), also referred to as bile acid receptor(“BAR”) or NR1H4 are known to be expressed in liver, intestine, kidneyand adrenal tissues. Activated FXR translocates to the cell nucleus andforms a heterodimer with retinoid X receptor (“RXR”). The dimer binds tohormone response elements on DNA causing, among other effects, asuppression of cholesterol 7 alpha-hydroxylase (“CYP7A1”), therate-limiting enzyme in bile acid synthesis from cholesterol, andstimulation of intestinal bile acid binding protein (“IBABP”). BothCYP7A1 and IBABP are involved in homeostasis of bile acid andcholesterol. Bile acids such as chenodeoxycholic acid (“CDCA”),lithocholic acid (“LCA”) and deoxycholic acid (“DCA”) are known to actas agonists of FXR. In one embodiment, the present invention provides amethod of inhibiting FXR (e.g., agonizing) in a mammal, the methodcomprising administering to mammal a pharmaceutical composition asdescribed herein. In some embodiments, the mammal has one or more of: anelevated baseline alanine aminotransferase (“ALT”) level, an elevatedbaseline aspartate aminotransferase (“AST”) level, liver fibrosis, anelevated baseline gamma-glutanyl transferase level, an elevated baselinealkaline phosphatase level, an elevated baseline antimitochondrialantibody level, an elevated baseline antinuclear antibody level, anelevated total serum bilirubin level, and an elevated transaminaselevel. In some embodiments, the present invention provides a method ofinhibiting FXR in a mammal, the method comprising co-administeringorally to the mammal about 2 g to about 4 g per day of a pharmaceuticalcomposition comprising at least about 90%, by weight of all fatty acidspresent, ethyl eicosapentaenoate and about 1 mg to about 500 mg ofobeticholic acid or derivative thereof. In some embodiments, the presentinvention provides a method of inhibiting FXR in a mammal, the methodcomprising co-administering orally to the mammal about 2 to about 4capsules per day, each capsule comprising about 900 mg to about 1 g ofethyl eicosapentaenoate and not more than about 20%, by weight of allfatty acids present, docosahexaenoic acid or its esters, and about 1 mgto about 500 mg of obeticholic acid or derivative thereof. In someembodiments, the ethyl eicosapentaenoate and the obeticholic acid orderivative thereof are formulated in a single dosage unit. In someembodiments, the mammal is a human.

In some embodiments, the subject also has a cardiovascular-relateddisease. The term “cardiovascular-related disease” herein refers to anydisease or disorder of the heart or blood vessels (i.e. arteries andveins) or any symptom thereof. Non-limiting examples ofcardiovascular-related disease and disorders includehypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia, coronaryheart disease, vascular disease, stroke, atherosclerosis, arrhythmia,hypertension, myocardial infarction, and other cardiovascular events.

In one embodiment, the present invention provides a method of bloodlipid therapy comprising administering to a subject or subject group inneed thereof a pharmaceutical composition as described herein. Inanother embodiment, the subject or subject group hashypertriglyceridemia, hypercholesterolemia, mixed dyslipidemia and/orvery high triglycerides.

In another embodiment, the subject or subject group being treated has abaseline triglyceride level (or median baseline triglyceride level inthe case of a subject group), fed or fasting, of at least about 300mg/dl, at least about 400 mg/dl, at least about 500 mg/dl, at leastabout 600 mg/dl, at least about 700 mg/dl, at least about 800 mg/dl, atleast about 900 mg/dl, at least about 1000 mg/dl, at least about 1100mg/dl, at least about 1200 mg/dl, at least about 1300 mg/dl, at leastabout 1400 mg/dl, or at least about 1500 mg/dl, for example about 400mg/dl to about 2500 mg/dl, about 450 mg/dl to about 2000 mg/dl or about500 mg/dl to about 1500 mg/dl.

In one embodiment, the subject or subject group being treated inaccordance with methods of the invention has previously been treatedwith another active agent (e.g., Lovaza®, ursodeoxycholic acid(Actigall®, Watson Pharma, Inc.) and/or obeticholic acid (INT-747,Intercept Pharmaceuticals)) and has experienced (a) an increase in, orno decrease in, LDL-C levels and/or non-HDL-C levels, (b) hepatocellularcarcinoma, (c) pruritus, (d) headache, and/or (e) constipation. In onesuch embodiment, therapy with the original active agent is discontinuedand replaced by a method of the present invention.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineabsolute plasma level of free EPA (or mean thereof in the case of asubject group) not greater than about 0.70 nmol/ml, not greater thanabout 0.65 nmol/ml, not greater than about 0.60 nmol/ml, not greaterthan about 0.55 nmol/ml, not greater than about 0.50 nmol/ml, notgreater than about 0.45 nmol/ml, or not greater than about 0.40 nmol/ml.In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a baseline fastingplasma level (or mean thereof) of free EPA, expressed as a percentage oftotal free fatty acid, of not more than about 3%, not more than about2.5%, not more than about 2%, not more than about 1.5%, not more thanabout 1%, not more than about 0.75%, not more than about 0.5%, not morethan about 0.25%, not more than about 0.2% or not more than about 0.15%.In one such embodiment, free plasma EPA and/or total fatty acid levelsare determined prior to initiating therapy.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineabsolute plasma level of total fatty acid (or mean thereof) not greaterthan about 250 nmol/ml, not greater than about 200 nmol/ml, not greaterthan about 150 nmol/ml, not greater than about 100 nmol/ml, or notgreater than about 50 nmol/ml.

In another embodiment, the subject or subject group being treated inaccordance with methods of the invention exhibits a fasting baselineplasma, serum or red blood cell membrane EPA level not greater thanabout 70 μg/ml, not greater than about 60 μg/ml, not greater than about50 μg/ml, not greater than about 40 μg/ml, not greater than about 30μg/ml, or not greater than about 25 μg/ml.

In another embodiment, methods of the present invention comprise a stepof measuring the subject's (or subject group's mean) baseline lipidprofile prior to initiating therapy. In another embodiment, methods ofthe invention comprise the step of identifying a subject or subjectgroup having one or more of the following: baseline non-HDL-C value ofabout 200 mg/dl to about 400 mg/dl, for example at least about 210mg/dl, at least about 220 mg/dl, at least about 230 mg/dl, at leastabout 240 mg/dl, at least about 250 mg/dl, at least about 260 mg/dl, atleast about 270 mg/dl, at least about 280 mg/dl, at least about 290mg/dl, or at least about 300 mg/dl; baseline total cholesterol value ofabout 250 mg/dl to about 400 mg/dl, for example at least about 260mg/dl, at least about 270 mg/dl, at least about 280 mg/dl or at leastabout 290 mg/dl; baseline vLDL-C value of about 140 mg/dl to about 200mg/dl, for example at least about 150 mg/dl, at least about 160 mg/dl,at least about 170 mg/dl, at least about 180 mg/dl or at least about 190mg/dl; baseline HDL-C value of about 10 to about 60 mg/dl, for examplenot more than about 40 mg/dl, not more than about 35 mg/dl, not morethan about 30 mg/dl, not more than about 25 mg/dl, not more than about20 mg/dl, or not more than about 15 mg/dl; and/or baseline LDL-C valueof about 50 to about 300 mg/dl, for example not less than about 100mg/dl, not less than about 90 mg/dl, not less than about 80 mg/dl, notless than about 70 mg/dl, not less than about 60 mg/dl or not less thanabout 50 mg/dl.

In a related embodiment, upon treatment in accordance with the presentinvention, for example over a period of about 1 to about 200 weeks,about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about50 weeks, about 1 to about 40 weeks, about Ito about 20 weeks, about Itoabout 15 weeks, about 1 to about 12 weeks, about Ito about 10 weeks,about Ito about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits one or more of the following outcomes:

(a) reduced triglyceride levels compared to baseline or placebo control;

(b) reduced Apo B levels compared to baseline or placebo control;

(c) increased HDL-C levels compared to baseline or placebo control;

(d) no increase in LDL-C levels compared to baseline or placebo control;

(e) a reduction in LDL-C levels compared to baseline or placebo control;

(f) a reduction in non-HDL-C levels compared to baseline or placebocontrol;

(g) a reduction in vLDL levels compared to baseline or placebo control;

(h) an increase in apo A-I levels compared to baseline or placebocontrol;

(i) an increase in apo A-I/apo B ratio compared to baseline or placebocontrol;

(j) a reduction in lipoprotein A levels compared to baseline or placebocontrol;

(k) a reduction in LDL particle number compared to baseline or placebocontrol;

(l) an increase in LDL size compared to baseline or placebo control;

(m) a reduction in remnant-like particle cholesterol compared tobaseline or placebo control;

(n) a reduction in oxidized LDL compared to baseline or placebo control;

(o) no change or a reduction in fasting plasma glucose (FPG) compared tobaseline or placebo control;

(p) a reduction in hemoglobin A_(1c) (HbA_(1c)) compared to baseline orplacebo control;

(q) a reduction in homeostasis model insulin resistance compared tobaseline or placebo control;

(r) a reduction in lipoprotein associated phospholipase A2 compared tobaseline or placebo control;

(s) a reduction in intracellular adhesion molecule-1 compared tobaseline or placebo control;

(t) a reduction in interleukin-6 compared to baseline or placebocontrol;

(u) a reduction in plasminogen activator inhibitor-1 compared tobaseline or placebo control;

(v) a reduction in high sensitivity C-reactive protein (hsCRP) comparedto baseline or placebo control;

(w) an increase in serum or plasma EPA compared to baseline or placebocontrol;

(x) an increase in red blood cell (RBC) membrane EPA compared tobaseline or placebo control;

(y) a reduction or increase in one or more of serum phospholipid and/orred blood cell content of docosahexaenoic acid (DHA), docosapentaenoicacid (DPA), arachidonic acid (AA), palmitic acid (PA), staeridonic acid(SA) or oleic acid (OA) compared to baseline or placebo control;

(z) a reduction or no increase in serum or plasma alanineaminotransferase (ALT) compared to baseline or placebo control;

(aa) a reduction or no increase in serum or plasma aspartateaminotransferase (AST) compared to baseline or placebo control;

(bb) a reduction or no increase in serum or plasma gamma-glutanyltranspeptidase (GGT) compared to baseline or placebo control;

(cc) a reduction in or no increase in serum or plasma alkalinephosphatase (AP) compared to baseline or placebo control;

(dd) a reduction or no increase in serum or plasma antimitochondrialantibody compared to baseline or placebo control;

(ee) a reduction or no increase in serum or plasma antinuclear antibodycompared to baseline or placebo control;

(ff) a reduction or no increase in serum or plasma conjugated bilirubincompared to baseline or placebo control;

(gg) prevention or reduction of liver scar tissue buildup and/or liverfibrosis compared to baseline or placebo control;

(hh) an increase in or no decrease in serum or plasma fibroblast growthfactor-19 (FGF-19) compared to baseline or placebo control; and/or

(ii) a reduction or no increase in serum or plasma immunoglobulin M(“IgM”) compared to baseline or placebo control.

In one embodiment, upon administering a composition of the invention toa subject, the subject exhibits a decrease in triglyceride levels, anincrease in the concentrations of EPA and DPA (n-3) in red blood cells,and an increase of the ratio of EPA:arachidonic acid in red blood cells.In a related embodiment the subject exhibits substantially no or noincrease in RBC DHA.

In one embodiment, methods of the present invention comprise measuringbaseline levels of one or more markers set forth in (a)-(ii) above priorto dosing the subject or subject group. In another embodiment, themethods comprise administering a composition as disclosed herein to thesubject after baseline levels of one or more markers set forth in(a)-(ii) are determined, and subsequently taking an additionalmeasurement of said one or more markers.

In another embodiment, upon treatment with a composition of the presentinvention, for example over a period of about 1 to about 200 weeks,about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about50 weeks, about 1 to about 40 weeks, about 1 to about 20 weeks, about 1to about 15 weeks, about 1 to about 12 weeks, about 1 to about 10 weeks,about 1 to about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits any 2 or more of, any 3 or more of,any 4 or more of, any 5 or more of, any 6 or more of, any 7 or more of,any 8 or more of, any 9 or more of, any 10 or more of, any 11 or moreof, any 12 or more of, any 13 or more of, any 14 or more of, any 15 ormore of, any 16 or more of, any 17 or more of, any 18 or more of, any 19or more of, any 20 or more of, any 21 or more of, any 22 or more of, any23 or more of, any 24 or more of, any 25 or more of, any 26 or more of,any 27 or more of, any 28 or more of, any 29 or more of, any 30 or moreof, any 31 or more of, any 32 or more of, any 33 or more of, any 34 ormore of, or all 35 of outcomes (a)-(ii) described immediately above.

In another embodiment, upon treatment with a composition of the presentinvention, the subject or subject group exhibits one or more of thefollowing outcomes:

(a) a reduction in triglyceride level of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) as compared to baseline or placebocontrol;

(b) a less than 30% increase, less than 20% increase, less than 10%increase, less than 5% increase or no increase in non-HDL-C levels or areduction in non-HDL-C levels of at least about 1%, at least about 3%,at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55% or at least about 75% (actual % change or median % change) ascompared to baseline or placebo control;

(c) substantially no change in HDL-C levels, no change in HDL-C levels,or an increase in HDL-C levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55% or at least about 75% (actual %change or median % change) as compared to baseline or placebo control;

(d) a less than 60% increase, a less than 50% increase, a less than 40%increase, a less than 30% increase, less than 20% increase, less than10% increase, less than 5% increase or no increase in LDL-C levels or areduction in LDL-C levels of at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 55% or at leastabout 75% (actual % change or median % change) as compared to baselineor placebo control;

(e) a decrease in Apo B levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, at least about 55% or at least about 75% (actual %change or median % change) as compared to baseline or placebo control;

(f) a reduction in vLDL levels of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,at least about 50%, or at least about 100% (actual % change or median %change) compared to baseline or placebo control;

(g) an increase in apo A-I levels of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline or placebo control;

(h) an increase in apo A-I/apo B ratio of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline or placebo control;

(i) a reduction in lipoprotein (a) levels of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline or placebo control;

(j) a reduction in mean LDL particle number of at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(k) an increase in mean LDL particle size of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, or at least about 100% (actual % changeor median % change) compared to baseline or placebo control;

(l) a reduction in remnant-like particle cholesterol of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(m) a reduction in oxidized LDL of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline or placebo control;

(n) substantially no change, no significant change, or a reduction (e.g.in the case of a diabetic subject) in fasting plasma glucose (FPG) of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline orplacebo control;

(o) substantially no change, no significant change or a reduction inhemoglobin A_(1c) (HbA_(1c)) of at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at leastabout 30%, at least about 35%, at least about 40%, at least about 45%,or at least about 50% (actual % change or median % change) compared tobaseline or placebo control;

(p) a reduction in homeostasis model index insulin resistance of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline orplacebo control;

(q) a reduction in lipoprotein associated phospholipase A2 of at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 45%, at least about 50%, or at least about 100%(actual % change or median % change) compared to baseline or placebocontrol;

(r) a reduction in intracellular adhesion molecule-1 of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(s) a reduction in interleukin-6 of at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, or at least about 100% (actual % change ormedian % change) compared to baseline or placebo control;

(t) a reduction in plasminogen activator inhibitor-1 of at least about5%, at least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(u) a reduction in high sensitivity C-reactive protein (hsCRP) of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, or at leastabout 100% (actual % change or median % change) compared to baseline orplacebo control;

(v) an increase in serum, plasma and/or RBC EPA of at least about 5%, atleast about 10%, at least about 15%, at least about 20%, at least about25%, at least about 30%, at least about 35%, at least about 40%, atleast about 45%, at least about 50%, at least about 100%, at least about200% or at least about 400% (actual % change or median % change)compared to baseline or placebo control;

(w) an increase in serum phospholipid and/or red blood cell membrane EPAof at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, r at least about 50%, at leastabout 100%, at least about 200%, or at least about 400% (actual % changeor median % change) compared to baseline or placebo control;

(x) a reduction or increase in one or more of serum phospholipid and/orred blood cell DHA, DPA, AA, PA and/or OA of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) compared to baseline or placebocontrol;

(y) a reduction in total cholesterol of at least about 5%, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55% or at least about 75%(actual % change or median % change) compared to baseline or placebocontrol;

(z) a reduction in ALT of at least about 5%, at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 37%, at least about 39%, atleast about 40%, at least about 41%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, or at least about 100%(actual % change or median % change) compared to baseline or placebocontrol;

(aa) a reduction in AST of at least about 1%, at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about 6%,at least about 7%, at least about 8%, at least about 9%, at least about10%, at least about 12%, at least about 14%, at least about 16%, atleast about 18%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, or atleast about 50% (actual % change or median % change) compared tobaseline or placebo control;

(bb) a reduction in GGT of at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 62%, at least about 65%, at least about 70%, at least about73%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or at least about 100% (actual %change or median % change) compared to baseline or placebo control;

(cc) a reduction in AP of at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 38%, at least about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, at least about 65%, at least about 70%, at least about75%, at least about 80%, at least about 85%, at least about 90%, atleast about 95%, or at least about 100% (actual % change or median %change) compared to baseline or placebo control;

(dd) a reduction in conjugated bilirubin of at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 100%, at least about 110%, at least about 120%, at leastabout 130%, at least about 138%, at least about 140%, at least about150%, at least about 160%, at least about 170%, at least about 180%, atleast about 188%, at least about 190%, or at least about 200% (actual %change or median % change) compared to baseline or placebo control;

(ee) an increase in FGF-19 of at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about100%, at least about 110%, at least about 111%, at least about 120%, atleast about 121%, at least about 130%, at least about 140%, at leastabout 150%, at least about 160%, at least about 170%, at least about180%, at least about 190%, at least about 200%, at least about 210%, atleast about 220%, at least about 230%, at least about 238%, at leastabout 240%, at least about 248%, at least about 250%, at least about260%, at least about 270%, at least about 280%, at least about 290%, atleast about 300% (actual % change or median % change) compared tobaseline or placebo control; and/or

(ff) a reduction in IgM of at least about 0.1 g/L, 0.2 g/L, at leastabout 0.3 g/L, at least about 0.4 g/L, at least about 0.5 g/L, at leastabout 0.6 g/L, at least about 0.7 g/L, at least about 0.8 g/L, at leastabout 0.9 g/L, at least about 1 g/L, at least about 1.1 g/L, at leastabout 1.2 g/L, at least about 1.3 g/L, at least about 1.4 g/L, or atleast about 1.5 g/L (actual or median change), or at least about 500%,at least about 600%, at least about 700%, at least about 800%, at leastabout 900%, at least about 1000%, at least about 1100%, at least about1200%, at least about 1300%, at least about 1400%, at least about 1500%(actual % change or median % change) compared to baseline or placebocontrol.

In one embodiment, methods of the present invention comprise measuringbaseline levels of one or more markers set forth in (a)-(ff) prior todosing the subject or subject group. In another embodiment, the methodscomprise administering a composition as disclosed herein to the subjectafter baseline levels of one or more markers set forth in (a)-(ff) aredetermined, and subsequently taking a second measurement of the one ormore markers as measured at baseline for comparison thereto.

In another embodiment, upon treatment with a composition of the presentinvention, for example over a period of about 1 to about 200 weeks,about 1 to about 100 weeks, about 1 to about 80 weeks, about 1 to about50 weeks, about 1 to about 40 weeks, about Ito about 20 weeks, about Itoabout 15 weeks, about 1 to about 12 weeks, about Ito about 10 weeks,about Ito about 5 weeks, about 1 to about 2 weeks or about 1 week, thesubject or subject group exhibits any 2 or more of, any 3 or more of,any 4 or more of, any 5 or more of, any 6 or more of, any 7 or more of,any 8 or more of, any 9 or more of, any 10 or more of, any 11 or moreof, any 12 or more of, any 13 or more of, any 14 or more of, any 15 ormore of, any 16 or more of, any 17 or more of, any 18 or more of, any 19or more of, any 20 or more of, any 21 or more of, any 22 or more of, any23 or more of, any 24 or more of, any 24 or more of, any 25 or more of,any 26 or more of, any 27 or more of, any 28 or more of, any 29 or moreof, any 30 or more of, or all 31 of outcomes (a)-(ff) describedimmediately above.

Parameters (a)-(ff) can be measured in accordance with any clinicallyacceptable methodology. For example, triglycerides, total cholesterol,HDL-C and fasting blood sugar can be sample from serum and analyzedusing standard photometry techniques. VLDL-TG, LDL-C and VLDL-C can becalculated or determined using serum lipoprotein fractionation bypreparative ultracentrifugation and subsequent quantitative analysis byrefractometry or by analytic ultracentrifugal methodology. Apo A1, Apo Band hsCRP can be determined from serum using standard nephelometrytechniques. Lipoprotein (a) can be determined from serum using standardturbidimetric immunoassay techniques. LDL particle number and particlesize can be determined using nuclear magnetic resonance (NMR)spectrometry. Remnants lipoproteins and LDL-phospholipase A2 can bedetermined from EDTA plasma or serum and serum, respectively, usingenzymatic immunoseparation techniques. Oxidized LDL, intercellularadhesion molecule-1 and interleukin-6 levels can be determined fromserum using standard enzyme immunoassay techniques. These techniques aredescribed in detail in standard textbooks, for example TietzFundamentals of Clinical Chemistry, 6^(th) Ed. (Burtis, Ashwood andBorter Eds.), WB Saunders Company.

In one embodiment, subjects fast for up to 12 hours prior to bloodsample collection, for example about 10 hours.

In another embodiment, the present invention provides a method oftreating or preventing NASH in a subject in need thereof, comprisingadministering or co-administering to the subject one or morecompositions as disclosed herein. In another embodiment, the presentinvention provides a method of treating or preventing NASH whilelowering triglycerides.

In another embodiment, the present invention provides a method oftreating or preventing PBC in a subject in need thereof, comprisingadministering or co-administering to the subject one or morecompositions as disclosed herein. In another embodiment, the presentinvention provides a method of treating or preventing PBC while loweringtriglycerides.

In another embodiment, the present invention provides a method oftreating or preventing primary hypercholesterolemia and/or mixeddyslipidemia (Fredrickson Types IIa and IIb) in a subject in needthereof, comprising administering to the subject one or morecompositions as disclosed herein. In a related embodiment, the presentinvention provides a method of reducing triglyceride levels in a subjector subjects when treatment with a statin or niacin extended-releasemonotherapy is considered inadequate (Frederickson type IVhyperlipidemia).

In another embodiment, the present invention provides a method oftreating or preventing risk of recurrent nonfatal myocardial infarctionin a subject with a history of myocardial infarction, comprisingadministering to the subject one or more compositions as disclosedherein.

In another embodiment, the present invention provides a method ofslowing progression of or promoting regression of atheroscleroticdisease in a subject in need thereof, comprising administering to asubject in need thereof one or more compositions as disclosed herein.

In another embodiment, the present invention provides a method oftreating or preventing very high serum triglyceride levels (e.g. TypesIV and V hyperlipidemia) in a subject in need thereof, comprisingadministering to the subject one or more compositions as disclosedherein.

In another embodiment, the present invention provides a method oftreating subjects having very high serum triglyceride levels (e.g.greater than 1000 mg/dl or greater than 2000 mg/dl) and that are at riskof developing pancreatitis, comprising administering to the subject oneor more compositions as disclosed herein.

In one embodiment, a composition of the invention is administered to asubject in an amount sufficient to provide a daily dose ofeicosapentaenoic acid of about 1 mg to about 10,000 mg, 25 about 5000mg, about 50 to about 3000 mg, about 75 mg to about 2500 mg, or about100 mg to about 1000 mg, for example about 1 mg, about 2 mg, about 3 mg,about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg,about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about35 mg, about 40 mg, about 45 mg, about 50 mg, about 75 mg, about 100 mg,about 125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg,about 250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg,about 375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg,about 500 mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg,about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg,about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg,about 875 mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg,about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1200 mg, about1225 mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg,about 1350 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450mg, about 1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about1575 mg, about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg,about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800mg, about 1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about1925 mg, about 1950 mg, about 1975 mg, about 2000 mg, about 2025 mg,about 2050 mg, about 2075 mg, about 2100 mg, about 2125 mg, about 2150mg, about 2175 mg, about 2200 mg, about 2225 mg, about 2250 mg, about2275 mg, about 2300 mg, about 2325 mg, about 2350 mg, about 2375 mg,about 2400 mg, about 2425 mg, about 2450 mg, about 2475 mg, about 2500mg, 2525 mg, about 2550 mg, about 2575 mg, about 2600 mg, about 2625 mg,about 2650 mg, about 2675 mg, about 2700 mg, about 2725 mg, about 2750mg, about 2775 mg, about 2800 mg, about 2825 mg, about 2850 mg, about2875 mg, about 2900 mg, about 2925 mg, about 2950 mg, about 2975 mg,about 3000 mg, about 3025 mg, about 3050 mg, about 3075 mg, about 3100mg, about 3125 mg, about 3150 mg, about 3175 mg, about 3200 mg, about3225 mg, about 3250 mg, about 3275 mg, about 3300 mg, about 3325 mg,about 3350 mg, about 3375 mg, about 3400 mg, about 3425 mg, about 3450mg, about 3475 mg, about 3500 mg, about 3525 mg, about 3550 mg, about3575 mg, about 3600 mg, about 3625 mg, about 3650 mg, about 3675 mg,about 3700 mg, about 3725 mg, about 3750 mg, about 3775 mg, about 3800mg, about 3825 mg, about 3850 mg, about 3875 mg, about 3900 mg, about3925 mg, about 3950 mg, about 3975 mg, about 4000 mg, about 4025 mg,about 4050 mg, about 4075 mg, about 4100 mg, about 4125 mg, about 4150mg, about 4175 mg, about 4200 mg, about 4225 mg, about 4250 mg, about4275 mg, about 4300 mg, about 4325 mg, about 4350 mg, about 4375 mg,about 4400 mg, about 4425 mg, about 4450 mg, about 4475 mg, about 4500mg, about 4525 mg, about 4550 mg, about 4575 mg, about 4600 mg, about4625 mg, about 4650 mg, about 4675 mg, about 4700 mg, about 4725 mg,about 4750 mg, about 4775 mg, about 4800 mg, about 4825 mg, about 4850mg, about 4875 mg, about 4900 mg, about 4925 mg, about 4950 mg, about4975 mg, about 5000 mg, about 5100 mg, about 5200 mg, about 5300 mg,about 5400 mg, about 5500 mg, about 5600 mg, about 5700 mg, about 5800mg, about 5900 mg, about 6000 mg, about 6100 mg, about 6200 mg, about6300 mg, about 6400 mg, about 6500 mg, about 6600 mg, about 6700 mg,about 6800 mg, about 6900 mg, about 7000 mg, about 7100 mg, about 7200mg, about 7300 mg, about 7400 mg, about 7500 mg, about 7600 mg, about7700 mg, about 7800 mg, about 7900 mg, about 8000 mg, about 8100 mg,about 8200 mg, about 8300 mg, about 8400 mg, about 8500 mg, about 8600mg, about 8700 mg, about 8800 mg, about 8900 mg, about 9000 mg, about9100 mg, about 9200 mg, about 9300 mg, about 9400 mg, about 9500 mg,about 9600 mg, about 9700 mg, about 9800 mg, about 9900 mg, or about10,000 mg.

In another embodiment, any of the methods disclosed herein are used intreatment or prevention of a subject or subjects that consume atraditional Western diet. In one embodiment, the methods of theinvention include a step of identifying a subject as a Western dietconsumer or prudent diet consumer and then treating the subject if thesubject is deemed a Western diet consumer. The term “Western diet”herein refers generally to a typical diet consisting of, by percentageof total calories, about 45% to about 50% carbohydrate, about 35% toabout 40% fat, and about 10% to about 15% protein. A Western diet mayalternately or additionally be characterized by relatively high intakesof red and processed meats, sweets, refined grains, and desserts, forexample more than 50%, more than 60% or more or 70% of total caloriescome from these sources.

In one embodiment, a composition for use in methods of the inventioncomprises eicosapentaenoic acid, or a pharmaceutically acceptable ester,derivative, conjugate or salt thereof, or mixtures of any of theforegoing, collectively referred to herein as “EPA.” The term“pharmaceutically acceptable” in the present context means that thesubstance in question does not produce unacceptable toxicity to thesubject or interaction with other components of the composition.

In one embodiment, the EPA comprises all-ciseicosa-5,8,11,14,17-pentaenoic acid. In another embodiment, the EPAcomprises an eicosapentaenoic acid ester. In another embodiment, the EPAcomprises a C₁-C₅ alkyl ester of eicosapentaenoic acid. In anotherembodiment, the EPA comprises eicosapentaenoic acid ethyl ester,eicosapentaenoic acid methyl ester, eicosapentaenoic acid propyl ester,or eicosapentaenoic acid butyl ester. In one embodiment, the EPAcomprises all-cis eicosa-5,8,11,14,17-pentaenoic acid ethyl ester.

In another embodiment, the EPA is in the form of ethyl-EPA, lithium EPA,mono-, di- or triglyceride EPA or any other ester or salt of EPA, or thefree acid form of EPA. The EPA may also be in the form of a2-substituted derivative or other derivative which slows down its rateof oxidation but does not otherwise change its biological action to anysubstantial degree.

In another embodiment, EPA is present in a composition useful inaccordance with methods of the invention in an amount of about 50 mg toabout 5000 mg, about 75 mg to about 2500 mg, or about 100 mg to about1000 mg, for example about 50 mg, about 75 mg, about 100 mg, about 125mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about 250mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about 375mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, about 500mg, about 525 mg, about 550 mg, about 575 mg, about 600 mg, about 625mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875mg, about 900 mg, about 925 mg, about 950 mg, about 975 mg, about 1000mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about1025 mg, about 1050 mg, about 1075 mg, about 1200 mg, about 1225 mg,about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about1475 mg, about 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg,about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about 1700mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg,about 1950 mg, about 1975 mg, about 2000 mg, about 2025 mg, about 2050mg, about 2075 mg, about 2100 mg, about 2125 mg, about 2150 mg, about2175 mg, about 2200 mg, about 2225 mg, about 2250 mg, about 2275 mg,about 2300 mg, about 2325 mg, about 2350 mg, about 2375 mg, about 2400mg, about 2425 mg, about 2450 mg, about 2475 mg, about 2500 mg, 2525 mg,about 2550 mg, about 2575 mg, about 2600 mg, about 2625 mg, about 2650mg, about 2675 mg, about 2700 mg, about 2725 mg, about 2750 mg, about2775 mg, about 2800 mg, about 2825 mg, about 2850 mg, about 2875 mg,about 2900 mg, about 2925 mg, about 2950 mg, about 2975 mg, about 3000mg, about 3025 mg, about 3050 mg, about 3075 mg, about 3100 mg, about3125 mg, about 3150 mg, about 3175 mg, about 3200 mg, about 3225 mg,about 3250 mg, about 3275 mg, about 3300 mg, about 3325 mg, about 3350mg, about 3375 mg, about 3400 mg, about 3425 mg, about 3450 mg, about3475 mg, about 3500 mg, about 3525 mg, about 3550 mg, about 3575 mg,about 3600 mg, about 3625 mg, about 3650 mg, about 3675 mg, about 3700mg, about 3725 mg, about 3750 mg, about 3775 mg, about 3800 mg, about3825 mg, about 3850 mg, about 3875 mg, about 3900 mg, about 3925 mg,about 3950 mg, about 3975 mg, about 4000 mg, 4025 mg, about 4050 mg,about 4075 mg, about 4100 mg, about 4125 mg, about 4150 mg, about 4175mg, about 4200 mg, about 4225 mg, about 4250 mg, about 4275 mg, about4300 mg, about 4325 mg, about 4350 mg, about 4375 mg, about 4400 mg,about 4425 mg, about 4450 mg, about 4475 mg, about 4500 mg, about 4525mg, about 4550 mg, about 4575 mg, about 4600 mg, about 4625 mg, about4650 mg, about 4675 mg, about 4700 mg, about 4725 mg, about 4750 mg,about 4775 mg, about 4800 mg, about 4825 mg, about 4850 mg, about 4875mg, about 4900 mg, about 4925 mg, about 4950 mg, about 4975 mg, or about5000 mg.

In one embodiment, a composition (or co-administration regimen) of theinvention comprises obeticholic acid or a derivative thereof.Obeticholic acid, also referred to as INT-747 (InterceptPharmaceuticals), 6-ECDCA, or 6α-ethyl chenodeoxycholic acid, has anIUPAC designation of (3α,5β,6α,7α)-6-ethyl-3,7-dihydroxy-cholan-24-oicacid, an empirical formula of C₂₆H₄₄O₄, and a molecular weight of 420.3g/mole. Its molecular structure is shown in formula (I), below:

As used herein, the term “OCA” refers to any one or more of obeticholicacid in its free acid form, an ester thereof, a salt thereof (e.g., apharmaceutically acceptable salt such as metallic salts made fromaluminum, calcium, lithium, magnesium, potassium, sodium, and/or zinc,or organic salts made from N,N′-dibenzylethylenediamme, chlorprocaine,choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine),and/or procaine), an ether thereof, an amide thereof, an amino acidconjugate thereof (e.g., a glycine conjugate and/or a taurineconjugate), a triglyceride thereof, a diglyceride thereof, or a solvatethereof.

In various embodiments, OCA is present in a composition useful inaccordance with methods of the invention in an amount of about 1 mg toabout 500 mg, about 2.5 mg to about 12.5 mg, about 5 mg to about 25 mg,or about 10 mg to about 50 mg, for example about 1 mg, about 2 mg, about3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about9 mg, about 10 mg, about 12 mg, about 12.5 mg, about 15 mg, about 20 mg,about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg,about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about125 mg, about 150 mg, about 175 mg, about 200 mg, about 225 mg, about250 mg, about 275 mg, about 300 mg, about 325 mg, about 350 mg, about375 mg, about 400 mg, about 425 mg, about 450 mg, about 475 mg, or about500 mg.

In any embodiment described herein, the OCA may be coadministered in aseparate dosage unit, or may be co-formulated with the ethyleicosapentaenoate. In some embodiments, the OCA is suspended in theethyl eicosapentaenoate, for example as a component of a fill within acapsule. In other embodiments, the OCA is a component of a coatingapplied to a dosage unit including the ethyl eicosapentaenoate. In otherembodiments, the OCA is in the form of microcapsules, microbeads,granules, a powder or an oil mixed with the ethyleicosapentaenoate.

Thus, in various embodiments, compositions of the present inventioncomprise about 50 mg to about 5000 mg of EPA and about 1 mg to about 500mg of OCA. In some embodiments, the composition comprises about 400 mgto about 600 mg of EPA (e.g., about 400 mg, about 450 mg, about 500 mg,about 550 mg, or about 600 mg) and about 2.5 to about 12.5 mg of OCA(e.g., about 2.5 mg, about 5 mg, about 7.5 mg, about 10 mg, or about12.5 mg). In some embodiments, the composition comprises about 900 mg toabout 1150 mg of EPA (e.g., about 900 mg, about 950 mg, about 1000 mg,about 1050 mg, about 1100 mg, or about 1150 mg) and about 2.5 mg toabout 25 mg of OCA (e.g., about 2.5 mg, about 5 mg, about 10 mg, about12.5 mg, about 15 mg, about 20 mg, or about 25 mg). In some embodiments,the composition comprises about 1850 mg to about 2150 mg of EPA (e.g.,1850 mg, about 1900 mg, about 1950 mg, about 2000 mg, about 2050 mg,about 2100 mg, or about 2150 mg) and about 5 mg to about 50 mg of OCA(e.g., 5 mg, about 10 mg, about 12.5 mg, about 15 mg, about 20 mg, about25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, or about 50mg). In some embodiments, the composition comprises about 3750 mg toabout 4250 mg of EPA (e.g., about 3750 mg, about 3800 mg, about 3850 mg,about 3900 mg, about 3950 mg, about 4000 mg, about 4050 mg, about 4100mg, about 4150 mg, about 4200 mg, or about 4250 mg) and about 10 mg toabout 50 mg of OCA (e.g., about 10 mg, about 12.5 mg, about 15 mg, about20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg,or about 50 mg).

In various embodiments, compositions of the present invention compriseEPA and OCA in a weight ratio of EPA to OCA of about 1:1 to about1000:1, about 2:1 to about 500:1, about 40:1 to about 400:1, or about80:1 to about 200:1, for example about 1:1, about 2:1, about 3:1, about4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1,about 15:1, about 20:1, about 25:1, about 30:1, about 35:1, about 40:1,about 45:1, about 50:1, about 55:1, about 60:1, about 65:1, about 70:1,about 75:1, about 80:1, about 85:1, about 90:1, about 95:1, about 100:1,about 125:1, about 150:1, about 175:1, about 200:1, about 225:1, about250:1, about 275:1, about 300:1, about 325:1, about 350:1, about 375:1,about 400:1, about 425:1, about 450:1, about 475:1, about 500:1, about525:1, about 550:1, about 575:1, about 600:1, about 625:1, about 650:1,about 675:1, about 700:1, about 725:1, about 750:1, about 775:1, about800:1, about 825:1, about 850:1, about 875:1, about 900:1, about 925:1,about 950:1, about 975:1, or about 1000:1.

In another embodiment, a composition useful in accordance with theinvention contains not more than about 20%, not more than about 19%, notmore than about 18%, not more than about 17%, not more than about 16%,not more than about 151%, not more than about 41%, not more than about31%, not more than about 21%, not more than about 11%, not more thanabout 10%, not more than about 9%, not more than about 8%, not more thanabout 7%, not more than about 6%, not more than about 5%, not more thanabout 4%, not more than about 3%, not more than about 2%, not more thanabout 1%, or not more than about 0.5%, by weight of all fatty acidspresent, docosahexaenoic acid (DHA), if any. In another embodiment, acomposition of the invention contains substantially no docosahexaenoicacid. In still another embodiment, a composition useful in the presentinvention contains no docosahexaenoic acid and/or derivative thereof.

In another embodiment, EPA comprises at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%, by weight, of all fatty acids presentin a composition that is useful in methods of the present invention.

In one embodiment, a composition of the invention comprises ultra-pureEPA. The term “ultra-pure” as used herein with respect to EPA refers toa composition comprising at least 95% by weight EPA (as the term “EPA”is defined and exemplified herein). Ultra-pure EPA comprises at least96% by weight EPA, at least 97% by weight EPA, or at least 98% by weightEPA, wherein the EPA is any form of EPA as set forth herein.

In another embodiment, a composition useful in accordance with methodsof the invention contains less than 10%, less than 9%, less than 8%,less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,less than 2%, less than 1%, less than 0.5% or less than 0.25%, by weightof the total composition or by weight of the total fatty acid content,of any fatty acid other than EPA. Illustrative examples of a “fatty acidother than EPA” include linolenic acid (LA), arachidonic acid (AA),docosahexaenoic acid (DHA), alpha-linolenic acid (ALA), stearadonic acid(STA), eicosatrienoic acid (ETA) and/or docosapentaenoic acid (DPA). Inanother embodiment, a composition useful in accordance with methods ofthe invention contains about 0.1% to about 4%, about 0.5% to about 3%,or about 1% to about 2%, by weight, of total fatty acids other than EPAand/or DHA.

In another embodiment, a composition useful in accordance with theinvention has one or more of the following features: (a)eicosapentaenoic acid ethyl ester represents at least about 96%, atleast about 97%, or at least about 98%, by weight, of all fatty acidspresent in the composition; (b) the composition contains not more thanabout 4%, not more than about 3%, or not more than about 2%, by weight,of total fatty acids other than eicosapentaenoic acid ethyl ester; (c)the composition contains not more than about 0.6%, not more than about0.5%, or not more than about 0.4% of any individual fatty acid otherthan eicosapentaenoic acid ethyl ester; (d) the composition has arefractive index (20° C.) of about 1 to about 2, about 1.2 to about 1.8or about 1.4 to about 1.5; (e) the composition has a specific gravity(20° C.) of about 0.8 to about 1.0, about 0.85 to about 0.95 or about0.9 to about 0.92; (e) the composition contains not more than about 20ppm, not more than about 15 ppm or not more than about 10 ppm heavymetals, (f) the composition contains not more than about 5 ppm, not morethan about 4 ppm, not more than about 3 ppm, or not more than about 2ppm arsenic, and/or (g) the composition has a peroxide value of not morethan about 5 meq/kg, not more than about 4 meq/kg, not more than about 3meq/kg, or not more than about 2 meq/kg.

In another embodiment, a composition useful in accordance with theinvention comprises, consists of or consists essentially of at least 95%by weight ethyl eicosapentaenoate (EPA-E), about 0.2% to about 0.5% byweight ethyl octadecatetraenoate (ODTA-E), about 0.05% to about 0.25% byweight ethyl nonadecapentaenoate (NDPA-E), about 0.2% to about 0.45% byweight ethyl arachidonate (AA-E), about 0.3% to about 0.5% by weightethyl eicosatetraenoate (ETA-E), and about 0.05% to about 0.32% ethylheneicosapentaenoate (HPA-E). In another embodiment, the composition ispresent in a capsule shell.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 95%,96% or 97%, by weight, ethyl eicosapentaenoate, about 0.2% to about 0.5%by weight ethyl octadecatetraenoate, about 0.05% to about 0.25% byweight ethyl nonadecapentaenoate, about 0.2% to about 0.45% by weightethyl arachidonate, about 0.3% to about 0.5% by weight ethyleicosatetraenoate, and about 0.05% to about 0.32% ethylheneicosapentaenoate. Optionally, the composition contains not more thanabout 0.06%, about 0.05%, or about 0.04%, by weight, DHA or derivativethereof such as ethyl-DHA. In one embodiment the composition containssubstantially no or no amount of DHA or derivative thereof such asethyl-DHA. The composition further optionally comprises one or moreantioxidants (e.g. tocopherol) or other impurities in an amount of notmore than about 0.5% or not more than 0.05%. In another embodiment, thecomposition comprises about 0.05% to about 0.4%, for example about 0.2%by weight tocopherol. In another embodiment, about 500 mg to about 1 gof the composition is provided in a capsule shell.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 96% byweight ethyl eicosapentaenoate, about 0.22% to about 0.4% by weightethyl octadecatetraenoate, about 0.075% to about 0.20% by weight ethylnonadecapentaenoate, about 0.25% to about 0.40% by weight ethylarachidonate, about 0.3% to about 0.4% by weight ethyl eicosatetraenoateand about 0.075% to about 0.25% ethyl heneicosapentaenoate. Optionally,the composition contains not more than about 0.06%, about 0.05%, orabout 0.04%, by weight, DHA or derivative thereof such as ethyl-DHA. Inone embodiment the composition contains substantially no or no amount ofDHA or derivative thereof such as ethyl-DHA. The composition furtheroptionally comprises one or more antioxidants (e.g. tocopherol) or otherimpurities in an amount of not more than about 0.5% or not more than0.05%. In another embodiment, the composition comprises about 0.05% toabout 0.4%, for example about 0.2% by weight tocopherol. In anotherembodiment, the invention provides a dosage unit comprising about 500 mgto about 1 g of the foregoing composition in a capsule shell. In oneembodiment, the dosage unit is a gel or liquid capsule and is packagedin blister packages of about 1 to about 20 capsules per sheet.

In another embodiment, compositions useful in accordance with theinvention comprise, consist essential of, or consist of at least 96%,97% or 98%, by weight of all fatty acids present, ethyleicosapentaenoate, about 0.25% to about 0.38% by weight of all fattyacids present ethyl octadecatetraenoate, about 0.10% to about 0.15% byweight of all fatty acids present ethyl nonadecapentaenoate, about 0.25%to about 0.35% by weight of all fatty acids present ethyl arachidonate,about 0.31% to about 0.38% by weight of all fatty acids present ethyleicosatetraenoate, and about 0.08% to about 0.20% by weight of all fattyacids present ethyl heneicosapentaenoate. Optionally, the compositioncontains not more than about 0.06%, about 0.05%, or about 0.04%, byweight of all fatty acids present, DHA or derivative thereof such asethyl-DHA. In one embodiment the composition contains substantially noor no amount of DHA or derivative thereof such as ethyl-DHA. Thecomposition further optionally comprises one or more antioxidants (e.g.tocopherol) or other impurities in an amount of not more than about 0.5%or not more than 0.05% by weight of all fatty acids present. In anotherembodiment, the composition comprises about 0.05% to about 0.4% byweight of all fatty acids present, for example about 0.2% by weight ofall fatty acids present, tocopherol. In another embodiment, theinvention provides a dosage unit comprising about 500 mg to about 1 g ofthe foregoing composition in a capsule shell along with about 1 mg toabout 50 mg of OCA.

In another embodiment, a composition as described herein is administeredto a subject once or twice per day. In another embodiment, 1, 2, 3 or 4capsules, each containing about 900 mg to about 1 g of a composition asdescribed herein, are administered to a subject daily. In anotherembodiment, 1 or 2 capsules, each containing about 900 mg to about 1 gof a composition as described herein, are administered to the subject inthe morning, for example between about 5 am and about 11 am, and 1 or 2capsules, each containing about 1 g of a composition as describedherein, are administered to the subject in the evening, for examplebetween about 5 pm and about 11 pm.

In one embodiment, a subject being treated in accordance with methods ofthe invention is not otherwise on lipid-altering therapy, for examplestatin, fibrate, niacin and/or ezetimibe therapy.

In another embodiment, compositions useful in accordance with methods ofthe invention are orally deliverable. The terms “orally deliverable” or“oral administration” herein include any form of delivery of atherapeutic agent or a composition thereof to a subject wherein theagent or composition is placed in the mouth of the subject, whether ornot the agent or composition is swallowed. Thus “oral administration”includes buccal and sublingual as well as esophageal administration. Inone embodiment, the composition is present in a capsule, for example asoft gelatin capsule.

A composition for use in accordance with the invention can be formulatedas one or more dosage units. The terms “dose unit” and “dosage unit”herein refer to a portion of a pharmaceutical composition that containsan amount of a therapeutic agent suitable for a single administration toprovide a therapeutic effect. Such dosage units may be administered oneto a plurality (i.e. 1 to about 10, 1 to 8, 1 to 6, 1 to 4 or 1 to 2) oftimes per day, or as many times as needed to elicit a therapeuticresponse.

In another embodiment, the invention provides use of any compositiondescribed herein for treating moderate to severe hypertriglyceridemia ina subject in need thereof, comprising: providing a subject having afasting baseline triglyceride level of about 500 mg/dl to about 1500mg/dl and administering to the subject a pharmaceutical composition asdescribed herein. In one embodiment, the composition comprises about 1 gto about 4 g of eicosapentaenoic acid ethyl ester, wherein thecomposition contains substantially no docosahexaenoic acid.

In one embodiment, compositions of the invention, upon storage in aclosed container maintained at room temperature, refrigerated (e.g.about 5 to about 5-10° C.) temperature, or frozen for a period of about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, exhibit at least about90%, at least about 95%, at least about 97.5%, or at least about 99% ofthe active ingredient(s) originally present therein.

In one embodiment, the invention provides use of a composition asdescribed herein in manufacture of a medicament for treatment of NASHand/or PBC and optionally a cardiovascular disease. In anotherembodiment, the subject is diabetic.

In one embodiment, a composition as set forth herein is packagedtogether with instructions for using the composition to treat and/orprevent NASH and/or PBC and optionally a cardiovascular disorder.

What is claimed is:
 1. A method of treating or preventing NASH in asubject, the method comprising co-administering orally to the subject apharmaceutical composition comprising at least about 80%, by weight ofall fatty acids present, eicosapentaenoic acid or a derivative thereof,less than about 10%, by weight of all fatty acids present,docosahexaenoic acid or its esters, and obeticholic acid or apharmaceutically acceptable salt, ester, glycine conjugate, or taurinederivative thereof.
 2. The method of claim 1, wherein the methodcomprises co-administering orally to the subject about 2 g to about 4 gper day of a pharmaceutical composition comprising and about 1 mg toabout 500 mg of obeticholic acid or a pharmaceutically acceptable salt,ester, glycine conjugate, taurine derivative thereof.
 3. The method ofclaim 1, wherein the method comprises co-administering orally to thesubject about 2 to about 4 capsules per day, each capsule comprisingabout 500 mg to about 1.1 g of ethyl eicosapentaenoate and not more thanabout 10% docosahexaenoic acid or its esters, by weight of all fattyacids present, and about 1 mg to about 500 mg of obeticholic acid or apharmaceutically acceptable salt, ester, glycine conjugate, taurinederivative thereof.
 4. The method of claim 1, wherein beforeadministration of the pharmaceutical composition, the subject or mammalhas one or more of: an elevated baseline alanine aminotransferase(“ALT”) level, an elevated baseline aspartate aminotransferase (“AST”)level, liver fibrosis, an elevated baseline gamma-glutanyl transferaselevel, an elevated baseline alkaline phosphatase level, an elevatedbaseline antimitochondrial antibody level, an elevated baselineantinuclear antibody level, an elevated total serum bilirubin level, andan elevated transaminase level.
 5. The method of claim 1, wherein afteradministration of the pharmaceutical composition for a period of time,the subject or mammal has one or more of: a reduced alanineaminotransferase (“ALT”) level, a reduced aspartate aminotransferase(“AST”) level, reduced liver fibrosis, a reduced gamma-glutanyltransferase level, a reduced alkaline phosphatase level, a reducedantimitochondrial antibody level, a reduced antinuclear antibody level,a reduced total serum bilirubin level, and a reduced transaminase levelcompared to baseline, to a second subject who has not received thepharmaceutical composition, or to placebo control.
 6. The method ofclaim 5, wherein a triglyceride level, an IgM level, and/or a C-reactiveprotein level is reduced in the subject compared to baseline, comparedto a second subject who has not received the pharmaceutical composition,or compared to placebo control.
 7. The method of claim 1, wherein thesubject is administered the pharmaceutical composition daily for aperiod of at least 2 weeks or at least 12 weeks.
 8. The method of claim1, wherein the pharmaceutical composition comprises at least about 85%,by weight of all fatty acids present, ethyl eicosapentaenoate.
 9. Themethod of claim 8, wherein the pharmaceutical composition comprises atleast about 90%, by weight of all fatty acids present, ethyleicosapentaenoate.
 10. The method of claim 1, wherein the pharmaceuticalcomposition comprises less than about 5%, by weight of all fatty acidspresent, docosahexaenoic acid or its esters.
 11. The method of claim 1,wherein the pharmaceutical composition comprises substantially nodocosahexaenoic acid or its esters.
 12. The method of claim 1, whereinthe subject is not administered an additional NASH.
 13. The method ofclaim 1, wherein the subject is administered an additional NASH.
 14. Themethod of claim 13, wherein the additional NASH therapeutic agent isselected from the group consisting of: L-alanine and pharmaceuticallyacceptable salts thereof ursodeoxycholic acid or a pharmaceuticallyacceptable derivative thereof;23-N-carbacinnamyloxy-3α,7α-dihydroxy-5β-norcholanylamine or apharmaceutically acceptable derivative thereof;

chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), lithocholic acid(LCA), and/or the taurine and/or glycine conjugates thereof; and organicor inorganic selenium, b-carotene and/or vitamin A.
 15. The method ofclaim 1, wherein the subject is administered about 4 g per day of thepharmaceutical composition.
 16. The method of claim 3, wherein thesubject is administered about 4 capsules per day.
 17. The method ofclaim 1, wherein the subject consumes a Western diet.